The Clotting Cascade
Point to note: Clotting factors were not discovered in numerical sequence hence the bizarre chain of events ensuing when clotting occurs. Hemostasis involves 3 steps Vascular spasms Platelet plug formation Coagulation. Spasm is the immediate response to blood vessel injury. Triggered by direct injury to vascular smooth muscle, by chemicals released by endothelial cells (e.g. damaged endothelial cells produce endothelin which is a vasoconstrictor) AND chemicals released from platelets (e.g serotonin). Neural reflexes initiated by local pain receptors also can induce spasm. Spasm is most effective in small blood vessels. It becomes more efficient as tissue damage increases. Gives time for platelet plug to form. Platelets normally don’t adhere to healthy endothelium. Damaged endothelial cells secrete Von Willebrand (a glycoprotein). This attaches to damaged endothelium and then attracts platelets floating by in the blood. VWF changes platelets, they become sticky, swell, form spiked processes and ATTACH to VWF. This process is called platelet adhesion and the platelets so attached become activated. Once active, the granules contained within the platelets break down to release several chemicals. Platelets have 2 types of granules. Alpha and Delta. The delta granules look like a sac and release SAC chemicals Serotonin, ADP and Calcium. Serotonin increases vascular spasm. ADP is a potent aggregating factor and attracts more platelets. These platelets stick to one another in a process called aggregation. Activated platelets also generate and release Thromboxane A2. This is vasoconstrictive and stimulates activation of new platelets as well as increasing platelet aggregation. Via a positive feedback mechanism, ever increasing numbers of platelets are drawn into the area and form a platelet plug. This is the primary hemostatic plug. The released Calcium from the delta granules functions to pin the coagulation factors down to the platelet surface to promote their activation and subsequent activity. Coagulation pathways are calcium dependent. The alpha granules in the platelets release some coagulation factors e.g. factor 5 and factor 8. They also have some fibrinogen and what’s called PLATELET DERIVED GROWTH FACTOR. This enters basement membrane of damaged blood vessel and promotes mitosis of smooth muscle. Fibroblasts will be attracted here and more collagen will be secreted to seal the damage. Coagulation is a property of plasma. Plasma by itself alone can coagulate. Coagulation is a process by which soluble fibrinogen converts into insoluble fibrin. It is a cascade of enzymatic reactions at the end of which soluble fibrinogen (protein normally produced by liver and found in blood plasma) is converted into insoluble fibrin strands. Blood coagulates via 2 pathways 1) Via the intrinsic pathway where it coagulates due to the activity of an intrinsic factor called factor XII (factor 12) as it comes into contact with non endothelial surfaces e.g. glass in test tubes or damaged collagen found below the endothelium of blood vessels 2) Via the Extrinsic pathway when blood comes into contact with tissue ‘juice’ from some damaged part of the body. This ‘juice’ contains Tissue Factor (Synonyms - TF or platelet tissue factor, factor III, or thrombokinase) and this Tissue factor promotes coagulation. When injuries occur BOTH pathways are activated. Blood coming into contact with sub endothelial surface adheres, initiating intrinsic pathway. TF is released from damaged cells and this initiates extrinsic pathway by activating factor 7. Clotting cascade. Blood coming into contact with sub endothelial surface or aggregated platelets activates clotting factor 12. This is converted into its active form 12 (a). Activated 12 (a) then activates factor 11 to its active form 11(a). Activated 11(a) then activates factor 9 changing it to active factor 9(a). Once factor 9(a) is in the presence of phospholipid (found on surface of platelet), together with factor 8, and calcium, activated 9(a) can now change factor 10 into its activated form , factor 10(a). Activated 10(a) can now act on PROTHROMBIN (factor 2), to convert it to THROMBIN. This process requires calcium, the presence of phospholipid, and factor 5. Thrombin is the superactive molecule that converts fibrinogen to fibrin monomers. Thrombin now activates factor 13, an enzyme also called Thrombin stabilizing factor, and this enzyme interacts with fibrin monomers to cross link them to produce a plug that forms the secondary hemostatic plug. This is the intrinsic pathway cascade. The extrinsic cascade also is occurring simultaneously. Tissue factor released from injured tissues convert factor 7 into active 7(a). Activated 7(a) has a direct activating effect on factor 10, converting it to its active form 10(a). Then the rest of the cascade occurs via the common pathway (pathway shared by intrinsic and extrinsic paths- see drawing above) A clot is not a permanent solution, it needs to be replaced by new tissue. Within 30 to 60 minutes, the clot is further stabilized by a platelet induced process called clot retraction. Platelets contain contractile proteins actin and myosin. They contract the same way muscle does. When they do, they pull on the surrounding fibrin strands, squeezing serum from the mass, compacting the clot and drawing the ruptured edges of the blood vessel closer together. Vessel healing takes place, assisted by platelet derived growth factor released from the alpha granules of the platelets. Plasminogen (formed in the liver) floating by in the blood, is incorporated into the newly formed clot. This becomes activated by Thrombin, by activated factor 12 (a) and by Tissue plasminogen factor. It is activated to its active form PLASMIN and this begins to breakdown the clot, starting about 2 days after it’s formation. This process continues over several days till the clot is dissolved and replaced by new, healthy tissue. Once clotting cascade begins, it continues till a clot is formed. Two homeostatic mechanisms prevent clots from becoming unnecessarily large. 1) swift removal of clotting factors and 2) Inhibition of clotting factors. For clotting to occur, activated procoagulants must reach critical levels. Clot formation in flowing blood is usually curbed because activated clotting factors are diluted and washed away. What else can happen to the newly formed plug? 1) can be dislodged by blood flow. Forms an embolus if it’s in the circulatory system. 2)TPA may be produced in excess and destabilize the newly formed secondary hemostatic plug. 3) Fibroblasts may come into the plug area and lay down collagen thus organizing the plug and keeping it buried. Healthy blood vessels lined with undamaged endothelium are antiplatelet, antithrombotic and anticoagulant. Healthy endothelial cells produce Nitric oxide and Prostacyclin (PGI2) which are vasodilatory on smooth muscle and when these are in the lumen of the blood vessel, they also prevent platelet aggregation. Healthy endothelial cells express HEPARIN which is a platform for Antithrombin III (produced in liver and floating in blood). This heparin/antithrombin III combination destroys THROMBIN, activated factor 10(a)and activated 9 (a). Healthy endothelial cells express Thrombomodulin which binds to thrombin and this in turn binds to protein C. This complex inactivates activated factor 5(a) and activated 8(a). Healthy endothelial cells also produce a substance called Tissue Plasminogen Activator: this can convert a protein coming from the liver called plasminogen into its active form Plasmin. Plasmin degrades fibrin into fibrinogen degradation products. It’s via these mechanisms that Healthy endothelial cells are described as being antiplatelet, antithrombotic and anticoagulant, and it is by these mechanisms, clot formation is RESTRICTED to the damaged area since the activated clotting factors are prevented from acting outside this area.